original arTiClE A Mechanistically Relevant Cytotoxicity Assay Based on the Detection of Cellular Green Fluorescent Protein Michael Halter,1 Jamie L. Almeida,2 Alessandro Tona,2 Kenneth D. tection and decontamination.3 Ricin is a toxin derived from the Cole,1 Anne L. Plant,1 and John T. Elliott1 plant Ricinus communis, which can be found all over the world and mass-produced with little effort. The ricin toxin inhibits protein 1Cell Systems Science Group/Biochemical Science Division, synthesis by first binding to cell surface galactose residues and then National Institute of Standards and Technology, Gaithersburg, entering the cell via endocytosis, inactivating ribosomes catalyti- Maryland. cally and irreversibly.4 Biochemical assays5 for ricin activity do not 2SAIC, Arlington, Virginia. sufficiently measure the multiple processes, namely, cell entry, cy- toplasmic transport, and ribosomal inactivation, involved in ricin intoxication. A convenient, cell-based assay that was specific to aBSTraCT the action of such a toxin would allow the biological activity of the Cell-based assays for measuring ribosome inhibition by proteins protein to be assessed and would report the sensitivity of the cell to such as the plant toxin ricin are important for characterizing de- the receptor-mediated endocytosis and cytoplasmic transport of the contamination strategies and developing detection technologies for toxin. In this study, we report the use of a stably transfected line of field use. We report here an assay for ricin that provides a response Vero cells that expressed green fluorescent protein (GFP) and mea- that is relevant to the mechanism of ricin activity and permits a sured the loss of cellular GFP as an indicator for protein synthesis much faster readout than commonly used assays for cytotoxicity. inhibition by the ribosome inhibitor ricin. The GFP construct used The assay relies on the response of an engineered reporter cell line here consisted of the sequence for destabilized enhanced GFP ligated that was produced by stably transfecting Vero cells to express green to the promoter sequence for cytomegalovirus (CMV). Under control fluorescent protein (GFP) under the control of a cytomegalovirus of the CMV promoter, GFP was constitutively expressed by the cells, promoter. The results of the GFP-based assay were compared with and because it contained a proteasome-targeting peptide sequence,6 assay results from three commercially available cytotoxicity assays. it was rapidly degraded with a half-life of approximately 2–3 h.7,8 The GFP assay reports a sensitive response to ricin after 6 h of Changes in the synthesis rate of GFP due to interference with ribo- treatment, whereas the other assays require a 24-h incubation. some activity had a rapid effect on decreasing the cellular GFP levels Unlike the other assays, monitoring cellular GFP on a per-cell basis in affected cells. allows detection of reduced ribosome activity before significant cell We compared this assay, based on quantitative imaging to measure death occurs, and the results are not convoluted by the numbers of GFP levels in cells over time, with the 3-(4,5-dimethylthiazol-2-yl)- cells being assayed. 2,5-diphenyltetrazolium bromide (MTT) assay, the MultiTox-FluorTM (MTox) multiplex cytotoxicity assay (Promega, Madison, WI), and the CellTiter-Glo® (CTG) luminescent cell viability assay (Promega), inTroduCTion which are based on measurements of an overall response from a well icin is a type 2 ribosome-inactivating protein belonging to or plate of cells. These whole-well assays are intended to report cel- the A-B family of toxins1,2 and is of particular interest be- lular enzyme activity, but their results are convoluted by the number cause of its potential use as a biological weapon and the of cells present.9–11 A loss of signal from the assay can be caused Rdearth of rapid and sensitive assays that could assist de- by either the reduction of activity of the particular enzyme detected ABBREVIATIONS: CHX, cycloheximide; CMV, cytomegalovirus; CTG, CellTiter-Glo® (Promega, Madison, WI); FBS, fetal bovine serum; GFP, green fluorescent protein; IC50, 50% in- hibitory concentration; MEM, Minimum Essential Medium; MTox, MultiTox-Fluor; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NA, numerical aperture. The accepted SI unit of concentration, mol/L, has been represented by the symbol M in order to conform to the conventions of this journal. DOI: 10.1089/adt.2009.192 © mary ann lIebert, Inc. • VOl. 7 nO. 4 • aUGUSt 2009 AssAy and Drug Development Technologies 1 ADT.7.4Halter.2009.192.indd 1 6/11/09 5:05:02 PM HALTER et al. by the assay or a reduction in the number of cells in the well after Cell Culture, Toxin Preparation, and Plate Preparation cell death occurs. Furthermore, these assays frequently must be The stably transfected, monoclonal Vero cells were then maintained performed after the cells die (i.e., 24 h after intoxication) because of in MEM-FBS medium and maintained in a humidified 5% CO2 bal- continued cell activity during the apoptotic process. The GFP assay, anced-air atmosphere at 37°C. For the GFP expression stability exper- on the other hand, is more specific to the mechanism of action of the iments, three separate T-25 flasks were maintained. Before measure- toxin because the assay reports more directly on ribosome activity.12 ment of the GFP intensity by flow cytometry, cells were removed from Therefore, the fluorescence readout is sensitive to the translation of tissue culture polystyrene flasks with 500 μl of a 0.25% trypsin/0.53 GFP, not to proteins that were previously made and remain in the mM EDTA solution (American Type Culture Collection). Trypsin activ- cell. The assay is also a more rapid indicator of toxin activity for ity was quenched by the addition of an equivalent volume of growth the same reason. medium (Dulbecco’s Modified Eagle’s Medium plus 10% FBS). Flow cytometric analysis of the cellular populations were per- formed with a Coulter Epics XL flow cytometer (Beckman Coulter, MaTErialS and METhodS Fullerton, CA), equipped with a 15 mW output laser, tuned to 488 Preparation of GFP-Expressing Vero Cell Line nm. The green fluorescence was collected after a 525/40 nm band The CMV/pd2EGFP-1 construct was obtained by ligating the pass filter. Samples were gated on forward scatter versus side scat- CMV promoter to the gene for a short half-life destabilized GFP. ter to exclude debris and clumps and to select cell populations of The sequence encoding the immediate early CMV promoter was re- interest. The GFP-expressing Vero cells and the non-transfected moved from the pQCXIP vector (Clontech Laboratories, Palo Alto, control Vero cells were analyzed separately, and 10,000 events were CA) using restriction enzymes AgeI and BglII and subcloned into collected for each sample. vector pd2EGFP-1 (Clontech Laboratories) containing a gene en- Before each GFP-based imaging assay for ribosome inhibition, the 4 coding a destabilized variant of enhanced GFP, with a half-life of GFP-expressing Vero cells were seeded at 1 × 10 cells per well in 80 2 h as reported by the manufacturer. Vero cells (CCL-81, Ameri- μl of the growth medium into 96-well imaging plates (catalog number can Type Culture Collection, Manassas, VA) were maintained in 353219, BD Falcon, Franklin Lakes, NJ). Ricin (catalog number L-1090, Minimum Essential Medium (MEM) Alpha (Invitrogen, Carlsbad, 5 mg/ml, Vector Laboratories, Burlingame, CA) was serially diluted in CA) supplemented with 10% fetal bovine serum (FBS) (Invitrogen), the MEM-FBS medium to achieve the desired concentrations. Cyclo- 100 units/ml penicillin, and 100 μg/ml streptomycin (MEM-FBS heximide (CHX) (Sigma, St. Louis, MO) was diluted in the MEM-FBS medium) and maintained in a humidified 5% CO2 balanced-air at- medium from a 100 mg/ml stock solution in dimethyl sulfoxide. The mosphere at 37°C. Cells were transfected with the construct us- next day, 20 μl of CHX or ricin (Vector Laboratories) solution was added ing an Amaxa (Gaithersburg, MD) electroporator using solution V to each of the wells such that the desired final concentrations were (proprietary solution, Amaxa) and program V-001 (cell type-spe- reached. The plates were then maintained in a humidified 5% CO2 cific program, Amaxa) according to the manufacturer’s protocol balanced-air atmosphere at 37°C. Ten minutes prior to imaging, 100 μl (Amaxa Inc.). In brief, 500,000 cells suspended in solution V were of a 4 μg/ml Hoechst 33342 solution was added to each well, resulting incubated with 2 μg of construct or positive control vector pro- in a final concentration of 2 μg/ml. Immediately before imaging, the vided by the manufacturer (Amaxa) or no DNA and electroporated wells were aspirated, and the medium was replaced with Dulbecco’s with program V-001. Twenty-four hours after transfection, cells phosphate-buffered saline containing glucose. For the kinetic assays, a were switched to growth medium containing 800–1,200 μg/ml ge- single plate was seeded for the CHX time points, and a single plate was neticin (Invitrogen) for selection of stable transfectants.13 Colonies seeded for the ricin time points. Either CHX or ricin was added to three were visualized under fluorescence microscopy and scored based wells of each plate at 10 h, 8 h, 6 h, 4 h, 2 h, and immediately prior to on degree of brightness and fluorescence homogeneity. Individual imaging. The final concentrations of CHX and ricin were 100 μg/ml clones were subjected to two rounds of selection using sterile clon- (360 μM) and 5 ng/ml (76 pM), respectively. ing cylinders and visualized under phase microscopy to ensure Ricin solutions were prepared in a Biosafety Level 2 laboratory.